This proposal is aimed at continuing Dr. Lee's genetic analysis of the S. aureus type 8 capsular polysaccharide (CP8). The aims are: 1) to study the regulation of CP8 gene expression in response to environmental factors; 2) to characterize the function of the cap8 genes; and 3) to define the role of the microcapsules in virulence. To study the regulation of CP8 genes, the PI will use deletion and mutagenesis analyses of gene fusions to define which promoter sequences are required for gene regulation. He will clone regulatory genes that respond to different environmental stimuli by a reverse genetic approach and by transposon mutagenesis. The cloned gene will be inactivated by allelic replacement to study the regulation. He will study how CP8 genes are regulated by agr, a global regulator. To study the functions of CP8 genes, he will use chemical mutants that have already been generated. Each gene will be correlated with function by analyzing the accumulated intermediate resulting from a specific gene mutation. The focus will be on genes involved in chain-length regulation, transport, and polymerization. To define the role of microcapsules in pathogenesis, isogenic deletion mutants will be tested in an in vitro phagocytic assays and by mouse lethality studies.